Methods for the modulation of neovascularization and/or the growth of collateral arteries and/or other arteries from preexisting arteriolar connections

ABSTRACT

Described is the modulation of the neovascularization and/or growth of collateral arteries and/or other arteries from preexisting arteriolar connections. Methods are provided for enhancing neovascularization and/or the growth of collateral arteries and/or other arteries from preexisting arteriolar connections comprising contacting organs, tissue or cells with a colony stimulating factor (CSF) or a nucleic acid molecule encoding said CSF. Furthermore, the use of a CSF or a nucleic acid molecule encoding said CSF for the preparation of pharmaceutical compositions for enhancing neovascularization and/or collateral growth of collateral arteries and/or other arteries from preexisting arteriolar connections is described. Also provided are methods for the treatment of tumors comprising contacting an organ, tissue or cells with an agent which suppresses neovascularization and/or the growth of collateral arteries and/or other arteries from preexisting arteriolar connections through the inhibition of the biological activity of CSFs. Described is further the use of an agent which suppresses neovascularization and/or the growth of collateral arteries and/or other arteries from preexisting arteriolar connections through inhibition of the biological activity of CSFs for the preparation of pharmaceutical compositions for the treatment of tumors.

This application is a continuation of U.S. application Ser. No.09/509,764, which is the U.S. national phase of InternationalApplication No. PCT/EP98/06233, filed Oct. 1, 1998.

The present invention relates generally to the modulation ofneovascularization and/or the growth of collateral arteries or otherarteries from preexisting arteriolar connections. In particular, thepresent invention provides a method for enhancing neovascularizationand/or the growth of collateral arteries and/or other arteries frompreexisting arteriolar connections comprising contacting an organ,tissue or cells with a colony stimulating factor (CSF) or a nucleic acidmolecule encoding said CSF. The present invention also relates to theuse of a CSF or a nucleic acid molecule encoding said CSF for thepreparation of pharmaceutical compositions for enhancingneovascularization and/or collateral growth of collateral arteriesand/or other arteries from preexisting arteriolar connections.Furthermore, the present invention relates to a method for the treatmentof tumors comprising contacting an organ, tissue or cells with an agentwhich suppresses neovascularization and/or the growth of collateralarteries and/or other arteries from preexisting arteriolar connectionsthrough the inhibition of the biological activity of a CSF. The presentinvention further involves the use of an agent which suppressesneovascularization and/or the growth of collateral arteries and/or otherarteries from preexisting arteriolar connections through the inhibitionof the biological activity of a CSF for the preparation ofpharmaceutical compositions for the treatment of tumors.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including any manufacturer'sspecifications, instructions, etc.) are hereby incorporated herein byreference; however, there is no admission that any document cited isindeed prior art as to the present invention.

In the treatment of subjects with arterial occlusive diseases most ofthe current treatment strategies aim at ameliorating their effects. Theonly curative approaches involve angioplasty (balloon dilatation) orbypassing surgery. The former carries a high risk of restenosis and canonly be performed in certain arterial occlusive diseases, like ischemicheart disease. The latter is invasive and also restricted to certainkinds of arterial occlusive diseases. There is no established treatmentfor the enhancement of neovascularization and/or collateral growth.

Vascular growth in adult organisms proceeds via two distinct mechanisms,sprouting of capillaries (angiogenesis) and in situ enlargement ofpreexisting arteriolar connections into true collateral arteries(Schaper, J. Collateral Circulation—Heart, Brain, Kidney, Limbs. Boston,Dordrecht, London: Kluwer Academic Publishers; 1993). Recent studieshave disclosed mechanisms leading to angiogenesis with vascularendothelial growth factor (VEGF) as a major component (Tuder, J. Clin.Invest. 95 (1995), 1798-1807; Plate, Nature 359 (1992), 845-848;Ferrara, Endocrine Reviews 13 (1992), 18-42; Klagsbrun, Annu. Rev.Physiol. 53 (1991), 217-239; Leung, Science 246 (1990), 1306-1309). Thisspecific endothelial mitogen is upregulated by hypoxia and is able topromote vessel growth when infused into rabbit hindlimbs after femoralartery excision (Takeshita, J. Clin. Invest. 93 (1994), 662-670;Bauters, Am. J. Physiol. 267 (1994), H1263-H1271). These studies howeverdid not distinguish between capillary sprouting, a mechanism calledangiogenesis, and true collateral artery growth. Whereas VEGF is onlymitogenic for endothelial cells, collateral artery growth requires theproliferation of endothelial and smooth muscle cells and pronouncedremodeling processes occur (Schaper, J. Collateral Circulation—Heart,Brain, Kidney, Limbs. Boston, Dordrecht, London: Kluwer AcademicPublishers; 1993; Jakeman, J. Clin. Invest. 89 (1992), 244-253; Peters,Proc. Natl. Acad. Sci. USA 90 (1993), 8915-8919; Millauer, Cell 72(1993), 835-846; Pasyk, Am. J. Physiol. 242 (1982), H1031-H1037).Furthermore mainly capillary sprouting is observed in ischemicterritories for example in the pig heart or in rapidly growing tumors(Schaper, J. Collateral Circulation—Heart, Brain, Kidney, Limbs. Boston,Dordrecht, London: Kluwer Academic Publishers; 1993; Plate, Nature 359(1992), 845-848; Bates, Curr. Opin. Genet. Dev. 6 (1996), 12-19; Bates,Curr. Opin. Genet. Dev. 6 (1996), 12-19; Gorge, Basic Res. Cardiol. 84(1989), 524-535). True collateral artery growth, however, is temporallyand spacially dissociated from ischemia in most models studied (Schaper,J. Collateral Circulation—Heart, Brain, Kidney, Limbs. Boston,Dordrecht, London: Kluwer Academic Publishers; 1993; Paskins-Hurlburt,Circ. Res. 70 (1992), 546-553). Other or additional mechanisms as thosedescribed for angiogenesis in ischemic territories are therefore neededto explain collateral artery growth. From previous studies it is knownthat these collateral arteries grow from preexisting arteriolarconnections (Schaper, J. Collateral Circulation—Heart, Brain, Kidney,Limbs. Boston, Dordrecht, London: Kluwer Academic Publishers; 1993).

However, while agents such as VEGF and other growth factors arepresently being employed to stimulate the development of angiogenesisafter arterial occlusion, such agents are not envisaged as being capableof modulating the growth of preexisting arteriolar connections into truecollateral arteries.

Thus, the technical problem of the present invention is to providepharmaceutical compositions and methods for the modulation ofneovascularization and/or the growth of collateral arteries and/or otherarteries from preexisting arteriolar connections.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the invention relates to a method for enhancing theneovascularization and/or the growth of collateral arteries and/or otherarteries from preexisting arteriolar connections comprising contactingan organ, tissue or cells with a colony stimulating factor (CSF) or anucleic acid molecule encoding said CSF.

The term “neovascularization” within the meaning of the presentinvention refers to a review of Sasayama, Circulation Res. 85 (1992),1197-1204.

For the purpose of the present invention the growth of arteries frompreexisting arteriolar connections is also called “arteriogenesis”. Inparticular, “arteriogenesis” is the in situ growth of arteries byproliferation of endothelial and smooth muscle cells from preexistingarteriolar connections supplying blood to ischemic tissue, tumor orsites of inflammation. These vessels largely grow outside the affectedtissue but are much more important for the delivery of nutrients to theischemic territory, the tumor or the site of inflammation thancapillaries sprouting in the diseased tissue by angiogenic processes.

In the context of the present invention the term “colony stimulatingfactor (CSF)” refers to proteins and peptides which can act onmacrophages and which are capable of promoting collateral artery growthby direct activation, proliferation and/or potentiation of the effectorfunctions of resident and newly-recruited macrophages. Thus, accordingto the present invention, any CSF or other substances which arefunctionally equivalent to a CSF, namely which are capable of promotingcollateral artery growth can be used for the purpose of the presentinvention. The action of the CSF employed in the present invention maynot be limited to the above-described specificity but they may also acton, for example eosinophils, lymphocyte subpopulations and/or stemcells. Advantageously, the CSF is antiatherogenic.

In accordance with the present invention, it has surprisingly been foundthat that locally appliedGranulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) caused asignificant increase in collateral artery growth. These results werebased on a marked increase of collateral conductance measurements.Peripheral pressures and collateral flows were measured under maximalvasodilation using Statham pressure transducers, fluorescentmicrospheres and FACS analysis which allowed the calculation ofcollateral conductances from pressure flow relations. Furthermore, postmortem angiograms revealed a significantly higher number of collateralarteries compared to untreated animals. To the best of the inventors'knowledge, this is the very first report that antiatherogenic and widelyclinical established colony stimulating factors are capable ofsignificantly enhancing neovascularization and/or collateral arterygrowth and/or the growth of other arteries from preexisting arteriolarconnections in vivo. Hence, CSFs that can be employed in accordance withthe present invention are particularly suited for the treatment ofatheriosclerosis.

Experiments performed within the scope of the present inventiondemonstrate that local infusion of GM-CSF increases both collateral- andperipheral conductance after femoral artery occlusion due to enhancedvessel growth by its proliferative effects on macrophages. Thus, CSFs ornucleic acid molecules encoding CSFs can be used for the activation andproliferation of macrophages which in turn leads to neovascularizationand/or the growth of collateral arteries as well as to growth ofarteries from preexisting arteriolar connections, which is needed forthe cure of several occlusive diseases. Granulocyte colony stimulatingfactor (G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF) belong to a family of glycoprotidic growth factors required forthe survival, growth and differentiation of heamatopoietic precursorcells. Therefore this substance has been used clinically to treatpatients with heamatologic and oncologic disorders. The action of theseCSF molecules was thought to be restricted to cells of theheamatopoietic origin (Demetri, Semin. Oncol. 19 (1992), 362-385;Lieschke, N. Engl. J. Med. 327 (1992), 28-35/Comments 99-106).Furthermore, several studies have demonstrated that these colonystimulating factors also play a major role in lipid metabolism.

Although recent experiments have shown that GM-CSF is able to directlypromote a number of macrophage and granulocyte effector functionsincluding cell survival (Selgas, Kidney International 50 (1996),2070-2078; Lopez, J. Clin. Invest. 78 (1986), 1220-1228; Eischen, J.Immunol. Meth. 147 (1991), 3408-3412; Vincent, Exp. Hematol. 20 (1992),17-23; Mangan, J. Immunol. 147 (1991), 3408-3412), activation,proliferation (Hoedemakers, Hepatology 13 (1994), 666-674; Matsushime,Japanese Journal of Clinical Hematology 36 (1995), 406-409);differentiation (Munn, Cancer Immunology, Immunotherapy 41 (1995),46-52), and migration of local tissue macrophages (Bussolini, Nature 337(1989), 471-473) it was not known that GM-CSF or other colonystimulating factors play a role in the development of collateralarteries and arteriogenesis.

The CSFs to be employed in the methods and uses of the present inventionmay be obtained from various sources described in the prior art; see,e.g., Gaertner, Bioconjugate Chemistry 3 (1992), 262-268; Dexter,European Journal of Cancer 30A (1994), 15-9; Rohde, Developments inBiological Standardization 83 (1994), 121-127; Lu, Protein Expression &Purification 4 (1993), 465-472; Itoh, Tanpakushitsu KakusanKoso—Protein, Nucleic Acid, Enzyme 35, 2620-2631. The potential exists,in the use of recombinant DNA technology, for the preparation of variousderivatives of colony stimulating factor (CSF) comprising a functionalpart thereof or proteins which are functionally equivalent to CSFs asdescribed above. In this context, as used throughout this specification“functional equivalent or “functional part” of an CSF means a proteinhaving part or all of the primary structural conformation of a CSFpossessing at least the biological property of promoting at least onemacrophage or granulocyte effector function mentioned above. Thefunctional part of said protein or the functionally equivalent proteinmay be a derivative of an CSF by way of amino acid deletion(s),substitution(s), insertion(s), addition(s) and/or replacement(s) of theamino acid sequence, for example by means of site directed mutagenesisof the underlying DNA. Recombinant DNA technology is well known to thoseskilled in the art and described, for example, in Sambrook et al.(Molecular cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y. (1989)). Modified CSFsare described, e.g., in Yamasaki, Journal of Biochemistry 115 (1994),814-819.

CSFs or functional parts thereof or proteins which are functionallyequivalent to CSFs, may be produced by known conventional chemicalsyntheses or recombinant techniques employing the amino acid and DNAsequences described in the prior art; see, e.g., EP-A-0 177 568; Han,Source Gene 175 (1996), 101-104; Kothari, Blood Cells, Molecules &Diseases 21 (1995), 192-200; Holloway, European Journal of Cancer 30A(1994), 2-6. For example, CSFs may be produced by culturing a suitablecell or cell line which has been transformed with a DNA sequenceencoding upon expression under the control of regulatory sequences a CSFor a functional part thereof or a protein which is functionallyequivalent to CSF. Suitable techniques for the production of recombinantproteins are described in, e.g., Sambrook, supra. Methods forconstructing CSFs and proteins as described above useful in the methodsand uses of the present invention by chemical synthetic means are alsoknown to those of skill in the art.

In another embodiment, the invention relates to the use of a colonystimulating factor (CSF) or a nucleic acid molecule encoding said CSFfor the preparation of a pharmaceutical composition for enhancingneovascularization and/or collateral growth of collateral arteriesand/or other arteries from preexisting arteriolar connections.

The pharmaceutical composition comprises at least one CSF as definedabove, and optionally a pharmaceutically acceptable carrier or exipient.Examples of suitable pharmaceutical carriers are well known in the artand include phosphate buffered saline solutions, water, emulsions, suchas oil/water emulsions, various types of wetting agents, sterilesolutions etc. Compositions comprising such carriers can be formulatedby conventional methods. The pharmaceutical compositions can beadministered to the subject at a suitable dose. The dosage regimen maybe determined by the attending physician considering the condition ofthe patient, the severity of the disease and other clinical factors.Administration of the suitable compositions may be effected by differentways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular,topical or intradermal administration. The dosage regimen will bedetermined by the attending physician and other clinical factors. As iswell known in the medical arts, dosages for any one patient depends uponmany factors, including the patient's size, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. Generally, the regimen as a regular administration of thepharmaceutical composition should be in the range of 1 μg to 10 mg unitsper day. If the regimen is a continuous infusion, it should also be inthe range of 1 μg to 10 mg units per kilogram of body weight per minute,respectively. Progress can be monitored by periodic assessment. Dosageswill vary but a preferred dosage for intravenous administration of DNAis from approximately 10⁶ to 10¹² copies of the DNA molecule. Thecompositions of the invention may be administered locally orsystemically. Administration will generally be parenterally, e.g.,intravenously; DNA may also be administered directly to the target site,e.g., by biolistic delivery to an internal or external target site or bycatheter to a site in an artery.

In a preferred embodiment, said CSF used in the methods and uses of theinvention is selected from the group consisting ofGranulocyte-Macrophage-Colony-Stimulating Factor (GM-CSF),Granulocyte-Colony-Stimulating Factor (G-CSF),Macrophage-Colony-Stimulating Factor (M-CSF), Colony-Stimulating Factor(CSF-I), functionally equivalent substances or functional derivativesthereof.

In a preferred embodiment, the methods and uses of the invention may beemployed for diseases caused by a vascular disease or a cardiac infarctor a stroke or for any disease where an increase of blood supply viacollaterals, arteries etc. is needed.

In a particularly preferred embodiment, the methods and uses of theinvention are designed to be applied to a subject suffering fromarteriosclerosis, a coronary artery disease, a cerebral occlusivedisease, a peripheral occlusive disease, a visceral occlusive disease,renal occlusive disease, a mesenterial arterial insufficiency or anophthamic or retenal occlusion or for any disease where atheroscleroticplaques in the vascular wall lead to an obstruction of the vesseldiameter.

In a further preferred embodiment, the methods and uses of the inventionare designed to be applied to a subject during or after exposure to anagent or radiation or surgical treatment which damage or destroyarteries.

In a preferred embodiment, the CSF used in the methods and uses of theinvention is a recombinant CSF. DNA sequences encoding CSFs which can beused in the methods and uses of the invention are described in the priorart; see, e.g., Holloway, European Journal of Cancer 30A (1994), 2-6 orreferences cited above. Moreover, DNA and amino acid sequences of CSFsare available in the Gene Bank database. As described above, methods forthe production of recombinant proteins are well-known to the personskilled in the art; see, e.g., Sambrook, supra.

In a further preferred embodiment, the method and the use of the presentinvention is designed to be applied in conjugation with a growth factor,preferably fibroblast growth factor or vascular endothelial growthfactor (VEGF). This embodiment is particularly suited for enhancing ofboth sprouting of capillaries (angiogenesis) and in situ enlargement ofpreexisting arteriolar connections into true collateral arteries.Pharmaceutical compositions comprising, for example, CSF such as GM-CSF,and a growth factor such as VEGF may be used for the treatment ofperipheral vascular diseases or coronary artery disease.

In another preferred embodiment, the method of the invention comprises

-   (a) obtaining cells, tissue or an organ from a subject;-   (b) introducing into said cells, tissue or organ a nucleic acid    molecule encoding and capable of expressing the CSF in vivo; and-   (c) reintroducing the cells, tissue or organ obtained in step (b)    into the same subject or a different subject.

It is envisaged by the present invention that the CSFs and the nucleicacid molecules encoding the CSFs are administered either alone or incombination, and optionally together with a pharmaceutically acceptablecarrier or exipient. Said nucleic acid molecules may be stablyintegrated into the genome of the cell or may be maintained in a formextrachromosomally, see, e.g., Calos, Trends Genet. 12 (1996), 463-466.On the other hand, viral vectors described in the prior art may be usedfor transfecting certain cells, tissues or organs.

Furthermore, it is possible to use a pharmaceutical composition of theinvention which comprises a nucleic acid molecule encoding a CSF in genetherapy. Suitable gene delivery systems may include liposomes,receptor-mediated delivery systems, naked DNA, and viral vectors such asherpes viruses, retroviruses, adenoviruses, and adeno-associatedviruses, among others. Delivery of nucleic acid molecules to a specificsite in the body for gene therapy may also be accomplished using abiolistic delivery system, such as that described by Williams (Proc.Natl. Acad. Sci. USA 88 (1991), 2726-2729).

Standard methods for transfecting cells with nucleic acid molecules arewell known to those skilled in the art of molecular biology, see, e.g.,WO 94/29469. Gene therapy to prevent or decrease the development ofdiseases described herein may be carried out by directly administeringthe nucleic acid molecule encoding a CSF to a patient or by transfectingcells with said nucleic acid molecule ex vivo and infusing thetransfected cells into the patient. Furthermore, research pertaining togene transfer into cells of the germ line is one of the fastest growingfields in reproductive biology. Gene therapy, which is based onintroducing therapeutic genes into cells by ex-vivo or in-vivotechniques is one of the most important applications of gene transfer.Suitable vectors and methods for in-vitro or in-vivo gene therapy aredescribed in the literature and are known to the person skilled in theart; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper,Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813;Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995),1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO94/29469; WO97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640,and references cited therein. The nucleic acid molecules comprised inthe pharmaceutical composition of the invention may be designed fordirect introduction or for introduction via liposomes, or viral vectors(e.g. adenoviral, retroviral) containing said nucleic acid molecule intothe cell. Preferably, said cell is a germ line cell, embryonic cell, oregg cell or derived therefrom.

It is to be understood that the introduced nucleic acid moleculesencoding the CSF express said CSF after introduction into said cell andpreferably remain in this status during the lifetime of said cell. Forexample, cell lines which stably express said CSF may be engineeredaccording to methods well known to those skilled in the art. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with the recombinant DNA molecule orvector of the invention and a selectable marker, either on the same orseparate vectors. Following the introduction of foreign DNA, engineeredcells may be allowed to grow for 1-2 days in an enriched media, and thenare switched to a selective media. The selectable marker in therecombinant plasmid confers resistance to the selection and allows forthe selection of cells having stably integrated the plasmid into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines which express a CSF. Such cells may be also beadministered in accordance with the pharmaceutical compositions, methodsand uses of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, Cell 11 (1977), 223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska, Proc. Natl.Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyltransferase(Lowy, Cell 22 (1980), 817) in tk⁻, hgprt⁻ or aprt⁻ cells, respectively.Also, antimetabolite resistance can be used as the basis of selectionfor dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl.Acad. Sci. USA 77 (1980), 3567; O'Hare, Proc. Natl. Acad. Sci. USA 78(1981), 1527), gpt, which confers resistance to mycophenolic acid(Mulligan, Proc. Natl. Acad. Sci. USA 78 (1981), 2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, J.Mol. Biol. 150 (1981), 1); hygro, which confers resistance to hygromycin(Santerre, Gene 30 (1984), 147); or puromycin (pat, puromycin N-acetyltransferase). Additional selectable genes have been described, forexample, trpB, which allows cells to utilize indole in place oftryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); and ODC(ornithine decarboxylase) which confers resistance to the ornithinedecarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue, 1987, In: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory ed.).

Thus, in a preferred embodiment, the nucleic acid molecule comprised inthe pharmaceutical composition for the use of the invention is designedfor the expression of the CSF by cells in vivo by, for example, directintroduction of said nucleic acid molecule or introduction of a plasmid,a plasmid in liposomes, or a viral vector (e.g. adenoviral, retroviral)containing said nucleic acid molecule.

In a preferred embodiment of the method and uses of the presentinvention, the CSF derivative or functional equivalent substance is anantibody, (poly)peptide, nucleic acid, small organic compound, ligand,hormone, PNA or peptidomimetic.

In this context, it is understood that the CSFs to be employed accordingto the present invention may be, e.g., modified by conventional methodsknown in the art. For example, it is possible to use fragments whichretain the biological activity of CSFs as described above, namely thecapability of promoting collateral artery growth. This further allowsthe construction of chimeric proteins and peptides wherein otherfunctional amino acid sequences may be either physically linked by,e.g., chemical means to the CSF or may be fused by recombinant DNAtechniques well known in the art. Furthermore, folding simulations andcomputer redesign of structural motifs of the CSFs or their receptorscan be performed using appropriate computer programs (Olszewski,Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995),675-679). Computer modeling of protein folding can be used for theconformational and energetic analysis of detailed receptor and proteinmodels (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp.Med. Biol. 376 (1995), 37-45). In particular, the appropriate programscan be used for the identification of interactive sites of the CSF andits receptor by computer assistant searches for complementary peptidesequences (Fassina, Immunomethods 5 (1994), 114-120). Furtherappropriate computer systems for the design of protein and peptides aredescribed in the prior art, for example in Berry, Biochem. Soc. Trans.22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987), 1-13;Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from theabove-described computer analysis can be used for, e.g., the preparationof peptidomimetics of the CSFs or fragments thereof. Such pseudopeptideanalogues of the natural amino acid sequence of the protein may veryefficiently mimic the parent protein or peptide (Benkirane, J. Biol.Chem. 271 (1996), 33218-33224). For example, incorporation of easilyavailable achiral Ω-amino acid residues into a CSF protein or a fragmentthereof results in the substitution of amide bonds by polymethyleneunits of an aliphatic chain, thereby providing a convenient strategy forconstructing a peptidomimetic (Banerjee, Biopolymers 39 (1996),769-777). Superactive peptidomimetic analogues of small peptide hormonesin other systems are described in the prior art (Zhang, Biochem.Biophys. Res. Commun. 224 (1996), 327-331). Appropriate peptidomimeticsof CSF may also be identified by the synthesis of peptidomimeticcombinatorial libraries through successive amide alkylation and testingthe resulting compounds, e.g., according to the methods described in theprior art. Methods for the generation and use of peptidomimeticcombinatorial libraries are described in the prior art, for example inOstresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg.Med. Chem. 4 (1996), 709-715. Furthermore, antibodies or fragmentsthereof may be employed which, e.g., upon binding to a CSF-receptormimic the biological activity of a CSF.

Furthermore, a three-dimensional and/or crystallographic structure ofthe CSF or of its receptor can be used for the design of peptidomimeticinhibitors of the biological activity of a CSF (Rose, Biochemistry 35(1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

As discussed above, neovascularization and the growth of arteries frompreexisting arteriolar connections is essential for the delivery ofnutrition to tumors. Thus, if the growth of said vessels to the tumorwould be suppressed suppression and/or inhibition of tumor growth is tobe expected.

Accordingly, the present invention also relates to a method for thetreatment of tumors comprising contacting an organ, tissue or cells withan agent which suppresses neovascularization and/or the growth ofcollateral arteries and/or other arteries from preexisting arteriolarconnections through the inhibition of the biological activity of a CSF.

Tumor Macrophages require specific growth factors, e.g., M-CSF/CSF-1,for their proliferation throughout the G1 phase of the cell cycle. Oncecells enter S phase, macrophages complete mitosis in the absence ofM-CSF/CSF-1. During the G1 phase, cyclin D (a cell cyclus regulator,that together with cyclin dependent kinase (cdk 4) promotes entry of thecell into M-phase (Alberts, Biology of the Cell (1989), Second Edition)is induced by M-CSF/CSF-1 stimulation. The enzymatic activity of cyclinD could be negatively regulated by recently reported inhibitory proteinsto determine the timing for entry into S phase in macrophages(Matsushime, Japanese Journal of Clinical Hematology 36 (1995),406-409).

It could be shown that among CSF-dependent macrophages especiallymonocytes as well as tissue specific macrophages (in the femalereproductive tract) seem to be dependent on CSF-1 for their furtherdifferentiation (Maito, Mol. Reprod. Dev. 46 (1997), 85-91). Beyond thisGM-CSF/M-CSF are essential for the macrophage survival. Thus, as itcould be demonstrated in accordance with the present invention that CSFspromote neovascularization and collateral artery growth withdrawal ofthese factors should result in inhibition or decrease ofneovascularization and/or collateral artery growth and, thus, in thesuppression of tumor growth. Agents which suppress neovascularizationand/or the growth of collateral arteries and/or other arteries frompreexisting arteriolar connections may be peptides, proteins, nucleicacids, antibodies, small organic compounds, hormones, neuraltransmitters, peptidomimics, or PNAs (Milner, Nature Medicine 1 (1995),879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198).For the preparation and application of such compounds, the personskilled in the art can use the methods known in the art, for examplethose referred to above.

The present invention further relates to the use of an agent whichsuppresses neovascularization and/or the growth of collateral arteriesand/or other arteries from preexisting arteriolar connections throughthe inhibition of the biological activity of a CSF for the preparationof a pharmaceutical composition for the treatment of tumors.

In a preferred embodiment, the agent used in the methods and uses of theinvention as described above inhibits the biological activity of a CSFand/or inhibits an intracellular signal or signal cascade comprisingMAPK and/or JNK/SAPK triggered in macrophages through the receptor forthe CSF. Various receptors of CSFs are described in the prior art, forexample in Chemokine Receptors. Immunology Today (1996), Suppl S: 26-27;Bendel, Leukemia & Lymphoma 25 (1997), 257-270; Perentesis, Leukemia &Lymphoma 25 (1997), 247-256; Bishay, Scandinavian Journal of Immunology43 (1996), 531-536; Kluck, Annals of Hematology 66 (1993), 15-20;Raivich, Journal of Neuroscience Research 30 (1991), 682-686 or in Wong,Cellular Immunology 123 (1989), 445-455.

In another preferred embodiment, said receptor is a CSF receptor. Saidreceptor or specific domains thereof which a responsible for triggeringa signal leading to collateral artery growth may be blocked or modulatedby methods described herein.

In a preferred embodiment, the agent used in the methods and uses of theinvention is a(n) antibody, (poly)peptide, nucleic acid, small organiccompound, ligand, hormone, PNA or peptidomimetic.

Nucleic acid molecules specifically hybridizing to CSF encoding genesand/or their regulatory sequences may be used for repression ofexpression of said gene, for example due to an antisense or triple helixeffect or they may be used for the construction of appropriate ribozymes(see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) whichspecifically cleave the (pre)-mRNA of a gene encoding a CSF. The nucleicand amino acid sequences encoding CSFs are known in the art anddescribed, for example, in Han, Source Gene 175 (1996), 101-104;Kothari, Blood Cells, Molecules & Diseases 21 (1995), 192-200 or inHolloway, European Journal of Cancer 30A (1994), 2-6. Selection ofappropriate target sites and corresponding ribozymes can be done asdescribed for example in Steinecke, Ribozymes, Methods in Cell Biology50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460.

Nucleic acids comprise DNA or RNA or hybrids thereof. Furthermore, saidnucleic acid may contain, for example, thioester bonds and/or nucleotideanalogues, commonly used in oligonucleotide anti-sense approaches. Saidmodifications may be useful for the stabilization of the nucleic acidmolecule against endo- and/or exonucleases in the cell. Furthermore, theso-called “peptide nucleic acid” (PNA) technique can be used for theinhibition of the expression of a gene encoding a CSF. For example, thebinding of PNAs to complementary as well as various single stranded RNAand DNA nucleic acid molecules can be systematically investigated using,e.g., thermal denaturation and BIAcore surface-interaction techniques(Jensen, Biochemistry 36 (1997), 5072-5077). The synthesis of PNAs canbe performed according to methods known in the art, for example, asdescribed in Koch, J. Pept. Res. 49 (1997), 80-88; Finn, Nucleic AcidsResearch 24 (1996), 3357-3363. Furthermore, folding simulations andcomputer redesign of structural motifs of the CSFs and their receptorscan be performed as described above to design drugs capable ofinhibiting the biological activity of CSFs.

Furthermore, antibodies may be employed specifically recognizing CSF ortheir receptors or parts, i.e. specific fragments or epitopes, of suchCSFs and receptors thereby inactivating the CSF or the CSF receptor.These antibodies can be monoclonal antibodies, polygonal antibodies orsynthetic antibodies as well as fragments of antibodies, such as Fab, Fvor scFv fragments etc. Antibodies or fragments thereof can be obtainedby using methods which are described, e.g., in Harlow and Lane“Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988or EP-B1 0 451 216 and references cited therein. For example, surfaceplasmon resonance as employed in the BIAcore system can be used toincrease the efficiency of phage antibodies which bind to an epitope ofthe CSF or its receptor (Schier, Human Antibodies Hybridomas 7 (1996),97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

Putative inhibitors which can be used in accordance with the presentinvention including peptides, proteins, nucleic acids, antibodies, smallorganic compounds, ligands, hormones, peptidomimetics, PNAs and the likecapable of inhibiting the biological activity of a CSF or its receptormay be identified according to the methods known in the art, for exampleas described in EP-A-0 403 506 or in the appended examples.

In a preferred embodiment, the agent which blocks the interaction of theCSF and its receptor is selected from the group consisting of

-   (i) an anti-CSF antibody and an anti-CSF-receptor antibody; and/or-   (ii) a non-stimulatory form of a CSF protein and a soluble form of a    CSF-receptor.

Such antibodies as well as inactive and soluble forms of CSFs and theirreceptors, respectively, are described in, e.g., Kogut, Inflammation 21(1997) or in Shimamura, Journal of Histochemistry & Cytochemistry 38(1990), 283-286 and can be obtained according to methods known in theart; see, e.g., supra.

In a preferred embodiment of the present invention, the agent isdesigned to be expressed in vascular cells or cells surroundingpreexisting arteriolar connections to a tumor.

In a preferred embodiment, methods and uses of the invention areemployed for the treatment of a tumor which is a vascular tumor,preferably selected from the group consisting of Colon Carcinoma,Sarcoma, Carcinoma in the breast, Carcinoma in the head/neck,Mesothelioma, Glioblastoma, Lymphoma and Meningeoma.

In a preferred embodiment, the pharmaceutical composition in the use ofthe invention is designed for administration by catheter intraarterial,intravenous, intraperitoneal or subcutenous routes. In the examples ofthe present invention the CSF protein was administered locally viaosmotic minipump.

These and other embodiments are disclosed or are obvious from andencompassed by the description and examples of the present invention.Further literature concerning any one of the methods, uses and compoundsto be employed in accordance with the present invention may be retrievedfrom public libraries, using for example electronic devices. For examplethe public database “Medline” may be utilized which is available onInternet, e.g. under http://www.ncbi.nlm.nih.gov/PubMed/medline.html.Further databases and addresses, such as http://www.ncbi.nlm.nih.gov/,http://www.infobiogen.fr/,http://www.fmi.ch/biology/research_tools.html, http://www.tigr.org/, areknown to the person skilled in the art and can also be obtained using,e.g., http://www.lycos.com. An overview of patent information inbiotechnology and a survey of relevant sources of patent informationuseful for retrospective searching and for current awareness is given inBerks, TIBTECH 12 (1994), 352-364.

The use and methods of the invention can be used for the treatment ofall kinds of diseases hitherto unknown as being related to or dependenton the modulation of neovascularization and/or the growth of collateralarteries and/or other arteries from preexisting arteriolar connections.The methods and uses of the present invention may be desirably employedin humans, although animal treatment is also encompassed by the methodsand uses described herein.

The figures show

FIG. 1: Angiography of the whole right leg of an animal treated withGM-CSF.

FIG. 2: Angiography of the whole right leg (A) and of the collateralcirculation (B) (without Os femoris) of an animal treated with GM-CSF.

FIG. 3: Angiography of the collateral circulation (without Os femoris)of an animal treated with GM-CSF.

FIG. 4: Angiography of the whole right leg of an animal treated withPBS.

FIG. 5: Angiography of the collateral circulation (without Os femoris)of an animal treated with PBS.

The examples illustrate the invention.

EXAMPLE 1 Femoral Artery Occlusion of Animals and Local Delivery ofAgents

The present study was performed with the permission of the State ofHessen, Regierungspräsidium Darmstadt, according to section 8 of theGerman Law for the Protection of Animals. It confirms with the Guide forthe Care und Use of Laboratory Animals published by the US NationalInstitut of Health (NIH Publication No. 85-23, revised 1985).

6 rabbits were subjected to 7 days of right femoral artery occlusion.They were randomly assigned to either receive GM-CSF (Novartis,Nuernberg, Germany) (2ML-2, Alza Corp; 3 μg in 2 mL PBS at a rate of 10μL/h) or PBS locally via osmotic minipump. For the initial implantationof the osmotic minipumps, the animals were anesthetized with anintramuscular injection of ketamine hydrochloride (40 to 80 mg/kg bodyweight) and xylazine (8 to 9 mg/kg body weight). Supplementary doses ofanesthetic (10% to 20% of the initial dose) were given intravenously asneeded. The surgical procedure was performed under sterile conditions.Femoral arteries were exposed and cannulated with a sterile polyethylenecatheter (inner diameter: 1 mm; outer diameter: 1.5 mm) pointingupstream, with the tip of the catheter positioned distal to thebranching of the arteria circemflexa femoris. The catheter itself wasconnected to the osmotic minipump (2ML-2, Alza Corp), which wasimplanted under the skin of the lower right abdomen. After that theanimals were outfitted with a specially designed body suit that allowedthem to move freely but prevented self-mutilation. The rabbits werehoused individually with free access to water and chow to securemobility. The body weights and body temperature in rabbits treated withGM-CSF did not significantly differ from those of control rabbits. Serumvalues of total protein, albumin, glutamic oxaloacetic transaminase, andglutamic pyruvic transaminase were not significantly changed by theGM-CSF treatment.

Seven days after implantation the animals were again anasthetized withan intramuscular injection of ketamine hydrochloride and xylazine fortracheostomy and artificial ventilation. Anesthesia was deepened withpentobarbital (12 mg/kg body weight per hour). The carotid artery wascannulated for continuous pressure monitoring. The arteria saphena magna(anterior tibial artery in humans and main arterial supply to the lowerlimb and foot in the rabbit) was exposed just above the ankle andcannulated with sterile polyethylene heparinized tubing (inner diameter0.58 mm; outer diameter 0.9 6 mm). They were connected to a StathamP23DC pressure transducer (Statham, Spectramed) for measurement ofperipheral pressures (PP). After heparinization with 5000 Units heparin,the left femoral artery was exposed and cannulated with sterilepolyethylene catheter (inner diameter: 1 mm; outer diameter: 1.5 mm) forthe microsphere reference sample. After cannulation of the abdominalaorta a shunt was installed to ensure oxygenated blood flow from thecarotid artery via the canula in the abdominal aorta into the right andleft legs. A flow probe was installed to measure total flow to bothhindlimbs.

EXAMPLE 2 Ex Vivo Pressure-flow Relations

Maximum vasodilation was achieved by injecting 20 mg papaverine (Sigma)to the shunt at a flow rate of 20 ml/min. After stabilization ofperipheral and central pressures both legs were perfused via fourdifferent pressure. Each pressure gradient was combined with a bolus ofmicrospheres.

Five different perfusion pressures (30, 40, 50, 60, 80 mmHg) weregenerated in vivo with a roller pump installed in the above mentionedshunt between carotid artery and abdominal aorta. Peripheral pressuresand collateral flows were measured under maximal vasodilation(papaverine) using Statham pressure transducers.

For each pressure level microspheres with a different fluorescent color(either crimson, scarlett, blue-green, red or blue) were injected intothe mixing chamber, which was installed in the carotid-abdominal aorticshunt.

The following muscles were dissected from the leg: Quadriceps, adductorlongus, adductor magnus, gastrocnemius, soleus, and peroneal muscles.Each muscle was divided into 3 three consecutive samples from theproximal to the distal end. The whole muscle and afterwards each samplewere weighed and cut to small pieces. The muscle sample were then placedloosely into 12 mm×75 mm polystyrene tubes (Becton Dickinson & Co.Lincoln Park, N.J.) and 3 ml of SDS solution [SDS solution (BoehringerMannheim Corp.): 1% SDS (Boehringer Mannheim Corp.), 0.5% sodium azide(Sigma Chemical Company, St. Louis, Mo.), and 0.8% Tween-80 (FisherScientific, Fairlawn, N.J.) in 50 millimolar pH 8 tris buffer (SigmaChemical Company, St. Louis, Mo.)], 30 μl proteinase K solution(Boehringer Mannheim Corp.) and 1 ml of microspheres as internalstandard was added (13.7 μm, Fluorescein Kit, Flow Cytometry Standards,Corp. San Juan, P.R.). Each tube was capped and secured in a shakingwater bath for 24-48 hours. The samples were then subsequently spinnedat 1000 g for 45 minutes, the supernatant was pipetted off and thepellet was resuspended in 1 ml PBS (pH 7.4). Before FACS analysis theprobes were rigorously shaken. The microspheres were counted using aflow cytometer (FACS-Calibur) equipped with a second laser and adetector for a fourth fluorescence. Flows for each sample werecalculated from the number of microspheres in the sample (m^(s)), therespective microspheres count in the reference sample (m^(rs)), theinternal standard in the sample (ISs), internal standard in thereference sample (IS rs), the weight of the reference sample (W) and thetime during which the reference sample was withdrawn using followingequation.

${{flow}\;\left\lbrack {{mg}\text{/}{ml}} \right\rbrack} = {\frac{{m^{S} \cdot I}\; S^{\Gamma\; S}}{I\;{S^{S} \cdot m^{\Gamma\; S}}} \cdot \frac{w}{t}}$

-   -   m^(s)=sample microsphere    -   IS^(rs)=internal standard reference sample    -   IS^(s)=internal standard sample    -   m^(rs)=microsphere reference sample    -   w=weight    -   t=time

In the present model, collateral arteries developing after femoralartery occlusion in typical corkscrew formation supply blood to thedistal adductor region and the lower leg. The systemic pressure [SP] andperipheral pressure [PP] was measured.

Venous pressure was equal to atmospheric pressure [AP] (zero in thepresent case). Since arterial resistances are much lower than collateraland peripheral resistances, they can be neglected. SP represent thepressure at the stem region of the collateral arteries. PP is thepressure at the reentry region and is identical to the pressure head ofthe circulation in lower leg; AP, the pressure at the venous end of theperipheral circulation. Collateral flow is equal to the sum of flow tothe tissue of the distal adductor plus the flow to the tissue of thelower leg. Collateral resistance was defined as pressure differencebetween SP and PP divided by the flow going to the distal adductor anthe lower leg. Peripheral resistance was defined as PP divided by flowto the lower leg, and bulk conductance was defined as SP divided by bulkflow recorded with the ultrasonic flow probe. The reciprocal values ofthese resistances represent collateral, peripheral, and bulkconductance. Because a positive pressure intercept is observed event atmaximal vasodilation, all conductances were calculated from the slope ofpressure-flow relations. Data are described as mean±SD. Differencesamong data were assessed using unpaired Student's t-test for intergroupcomparisons and Mann-Whithney rank-sum test for unequal variances.Values of p≦0.05 were required for assumption of statisticalsignificance. Collateral conductance was significantly higher after 1week of occlusion in animals treated with GM-CSF compared with animalswithout this treatment.

TABLE 1 collateral conductance [ml/min/100 mmHg]

EXAMPLE 3 Post Mortem Angiography

Legs were perfused with Krebs-Henseleit buffered saline in a warmedwaterbath of 37° C. for 1 minute at a pressure of 80 mmHg, followed byperfusion with contrast medium (8 to 10 minutes at 80 mmHg) based onbismuth and gelatin according to a formula developed by Fulton (Fulton:The Coronary Arteries, Thomas Books, 1965). Subsequently, the contrastmedium was allowed to gel by placing the limbs on crushed ice for 45minutes. Angiograms were taken at two different angles in a Balteauradiography apparatus (Machlett Laboratories) using a single-envelopedStructurix D7DW film (AGFA). The resulting stereoscopic pictures allowedanalysis of collateral growth in three dimensions.

To differentiate between collateral vessels and muscle vessels forfurther quantification, Longland's definition of collateral arteries wasused (Longland et.al. 1954 “Description of collateral arteries” Verlag:Thomas). Stem, midzone and re-entry were identified under stereoscopicviewing using a 3-fold magnification of our angiograms. Collateralarteries then were divided in two groups: group one consisted of vesselswhose stem branched from the Arteria circumflexa femoris lateralis.Group two of the arteries originated from the Arteria profunda femoris.The length of the midzone in each group was almost the same, so theirmeasurement did not give any further information. Re-entry of thecollaterals from the first group usually descended into the Arteriagenus descendens, the second group into the Arteria caudalis femoris.Only about 10% of the collateral arteries originate from other vessels,e.g from the A. iliaca externa or from the A. iliaca interna.

Collateral vessels were marked after counting to make sure, that novessel was counted twice. A further 3-fold magnification was used tomeasure the diameter of the vessels with an accuracy of 0.1 mm.Postmortem angiograms exhibited corkscrew collaterals mainly in theadductor longus, adducotr magnus, and vasuts intermedius connecting theperusion bed of the arteria femoralis profunda to that of the arteriasaphene parva int the adductor muscles and the perfusion bed of thearteria circumflexa femoris lateralis to that of the arteriae genualesin the quadriceps muscle. Angiograms taken from hindlimsb of animalstreated with GM-CSF show a remarkable increase the diameter and densityof these collateral vessels. (Table 2, FIGS. 1 to 5)

TABLE 2 collateral arteries

The results of the experiments performed in accordance with the presentinvention indicate that CSFs are capable of mediating neovascularizationand/or collateral artery growth and/or growth of arteries frompreexisting arteriolar connections due to macrophage recruitment thatmight be mediated by a direct effect of CSFs on macrophage activation,proliferation, motility, and survival and, secondarily, bychemoattractant molecules released in response to locally administeredCSFs. Thus, the present invention provides for novel means and methodsfor the treatment of diseases which depend on neovascularization and/orcollateral artery growth.

The present invention is not to be limited in scope by its specificembodiments described which are intended as single illustrations ofindividual aspects of the invention and any proteins, nucleic acidmolecules, or compounds which are functionally equivalent are within thescope of the invention. Indeed, various modifications of the inventionin addition to those shown and described therein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Said modifications intended to fall within the scope of theappended claims. Accordingly, having thus described in detail preferredembodiments of the present invention, it is to be understood that theinvention defined by the appended claims is not to be limited toparticular details set forth in the above description as many apparentvariations thereof are possible without departing from the spirit orscope of the present invention.

1. A method of treating a stroke in a human subject by enhancing thegrowth of arteries by proliferation of endothelial and smooth musclecells from preexisting arteriolar connections of said subject, saidmethod comprising identifying a human subject with a stroke,administering to said subject a pharmaceutical composition comprising aG-CSF protein and a pharmaceutically acceptable carrier or excipient inan amount effective to enhance the growth of said arteries of saidsubject, thereby treating said stroke of said subject, wherein saidG-CSF is the only agent administered in said method of treating thatenhances the growth of said arteries.
 2. The method of claim 1, whereinsaid pharmaceutical composition consists of a G-CSF protein and apharmaceutically acceptable carrier or excipient.
 3. The method of claim1, wherein said pharmaceutical composition is administered by anintracoronary, intramuscular, intrarterial, intravenous,intraperitoneal, or subcutaneous route.
 4. The method of claim 3,wherein said G-CSF protein is administered by an intravenous route. 5.The method of claim 2, wherein said pharmaceutical composition isadministered by an intracoronary, intramuscular, intrarterial,intravenous, intraperitoneal, or subcutaneous route.
 6. The method ofclaim 5, wherein said G-CSF protein is administered by an intravenousroute.
 7. The method of claim 1, wherein said G-CSF is a recombinantG-CSF.
 8. A method of treating a stroke in a human subject by enhancingthe growth of arteries by proliferation of endothelial and smooth musclecells from preexisting arteriolar connections of said subject, saidmethod comprising identifying a human subject with a stroke,administering to said subject a pharmaceutical composition consistingof: a G-CSF protein, a growth factor, and a pharmaceutically acceptablecarrier or excipient, in an amount effective to enhance the growth ofsaid arteries of said subject, thereby treating said stroke of saidsubject wherein said G-CSF is the only agent administered in said methodof treating that enhances the growth of said arteries.